The ubiquitin proteasome pathway is the predominant biological mechanism of myofibrillar protein (MF) degradation.  To test the hypothesis that amino acid and insulin act synergistically to regulate proteolysis, two experimental models were employed;  an <i>in vivo </i>study on growing calves and an <i>in vitro</i> C2C12 myotubes culture. Calves growing at 0.3kg/day, were constantly infused with glucose at a low (LDG) or high (HDG) dose (to stimulate insulin) with or without essential amino acids (EAA).  Glucose infusions increased plasma insulin and IGF-1 concentrations in a dose dependent manner (P<0.05).  HDG was associated with decreased plasma urea nitrogen and 3-MH concentrations and 3-MH:creatinine output (an index of MF degradation) (P < 0.05).  Glucose infusions down regulated the expression of 14-kDa E2 ubiquitin conjugating enzyme and C2 20 S proteasome sub unit, however EAA did not alter the effect of raised plasma insulin on muscle ubiquitin proteasome pathway suggesting that under the conditions employed, EAA do not act synergistically with insulin to decrease myofibrillar protein degradation, <i>in vivo.</i> In the <i>in vitro</i> experiments, amino acid deprivation (0.2 X physiological concentration amino acid;  PC AA) of myotubes for 8 h was associated with increased (P < 0.05) proteolysis (measured from TCA soluble ³H-tyrosine release in the medium), compared to controls (1.0 X PC AA).  Addition of insulin inhibited this increase (P < 0.05).  Rapamycin significantly increased proteolysis in 1.0 X PC AA media suggesting amino acid might regulate proteolysis through mTOR signalling pathway.  Reduced amino acid supply also increased 14-kDa E2 and C2 mRNA expression compared to controls (P < 0.05).  Increasing leucine concentration in 0.2 X PC AA basal media showed a dose dependent decrease in protein degradation and expression of 14-kDa E2, in the presence of insulin.  In conclusion, the results suggested that decreased availability of amino acids was associated with increased total proteolysis and that anti-catabolic effect of amino acid in C2C12 muscle cell cultures, was additive to that of insulin.

Gelation properties of a salt extracted pea (Pisum sativum) protein isolate (PPIs) were evaluated with a goal of using this isolate as a meat extender. Microbial transglutaminase (MTG) was used to improve gelation of PPIs, muscle protein isolate (MPI) from chicken breast and the two combined. Gelation properties were evaluated using small amplitude oscillatory rheology and texture analysis. SDS-PAGE and differential scanning calorimetry were used to examine protein structure. Minimum gelation concentration for PPIs was 5%, lower than the 14% obtained for a commercial pea protein isolate (PPIc), possibly because the PPIc undergone denaturation whereas PPIs had not. Storage modulus (G') and loss modulus (G") increased with protein concentration and maximum gel strength for PPIs occurred at pH 4.0 in 0.3M NaCl. Higher or lower pH values affected protein charge and the potential for network formation. Higher salt concentrations resulted in increased denaturation temperatures, to a point where the proteins did not denature at the 95ºC temperature used for gel formation. When both heating and cooling rate were increased, gel strength decreased, though the cooling rate had a greater impact. Chaotropic salts enhanced gel strength, whereas non-chaotropic salts stabilized protein structure and decreased gel formation. Based on effects of guanidine hydrochloride, urea, propylene glycol, β-mercaptoethanol, dithiothreitol and N-ethylmaleimide, hydrophobic and electrostatic interaction and hydrogen bonds were involved in pea protein gel formation but disulfide bond contribution was minimal. Gels formed with MPI at concentrations as low as 0.5% and were strongest at 95ºC, higher than the ~ 65ºC normally used in meat processing. Good gels were formed at pH 6 with 0.6 to 1.2 M NaCl. Addition of MTG increased gel strength for PPIs, MPI, and a combination of the two. SDS-PAGE showed that bands in the 35~100kDa range became fainter with higher MTG levels but no new bands were found to provide direct evidence of interaction between muscle and pea proteins. Improved gel strength for the MPI/PPI mixture (3:1) containing MTG suggested that some crosslinking occurred. Higher heating temperatures and MTG addition led to the formation of MPI/PPI gel and demonstrated the potential for utilization of pea protein in muscle foods.

Gelation properties of a salt extracted pea (Pisum sativum) protein isolate (PPIs) were evaluated with a goal of using this isolate as a meat extender. Microbial transglutaminase (MTG) was used to improve gelation of PPIs, muscle protein isolate (MPI) from chicken breast and the two combined. Gelation properties were evaluated using small amplitude oscillatory rheology and texture analysis. SDS-PAGE and differential scanning calorimetry were used to examine protein structure. Minimum gelation concentration for PPIs was 5%, lower than the 14% obtained for a commercial pea protein isolate (PPIc), possibly because the PPIc undergone denaturation whereas PPIs had not. Storage modulus (G') and loss modulus (G") increased with protein concentration and maximum gel strength for PPIs occurred at pH 4.0 in 0.3M NaCl. Higher or lower pH values affected protein charge and the potential for network formation. Higher salt concentrations resulted in increased denaturation temperatures, to a point where the proteins did not denature at the 95ºC temperature used for gel formation. When both heating and cooling rate were increased, gel strength decreased, though the cooling rate had a greater impact. Chaotropic salts enhanced gel strength, whereas non-chaotropic salts stabilized protein structure and decreased gel formation. Based on effects of guanidine hydrochloride, urea, propylene glycol, β-mercaptoethanol, dithiothreitol and N-ethylmaleimide, hydrophobic and electrostatic interaction and hydrogen bonds were involved in pea protein gel formation but disulfide bond contribution was minimal. Gels formed with MPI at concentrations as low as 0.5% and were strongest at 95ºC, higher than the ~ 65ºC normally used in meat processing. Good gels were formed at pH 6 with 0.6 to 1.2 M NaCl. Addition of MTG increased gel strength for PPIs, MPI, and a combination of the two. SDS-PAGE showed that bands in the 35~100kDa range became fainter with higher MTG levels but no new bands were found to provide direct evidence of interaction between muscle and pea proteins. Improved gel strength for the MPI/PPI mixture (3:1) containing MTG suggested that some crosslinking occurred. Higher heating temperatures and MTG addition led to the formation of MPI/PPI gel and demonstrated the potential for utilization of pea protein in muscle foods.

This study investigated the feasibility of oxidative modification with glucose oxidase (GluOx) to enhance the rheological and gelling properties of myofibrillar protein. Differential oxidative modifications of myofibrillar protein (MP) by hydroxyl radicals generated in an enzymatic system with glucose oxidase (GluOx) in the presence of glucose/FeSO4 compared to a Fenton system (H2O2/FeSO4) were investigated. Firmer and more elastic MP gels were produced by the GluOx-oxidizing system than by the Fenton system at comparable H2O2 levels due to an altered radical reaction pathway. The study further explored the effect of GluOx-mediated oxidation on the efficacy of transglutaminase (TGase) cross-linking of MP in 0.6 M NaCl and the rheological properties of GluOx oxidation/TGase treated MP in MP–lipid emulsion composite gels. The GluOx-mediated oxidation promoted the formation of both soluble and insoluble protein aggregates via disulfide bonds and occlusions of hydrophobic groups. The subsequent TGase treatment converted protein aggregates into highly cross-linked polymers. MP–lipid emulsion composite gels formed with such polymers exhibited markedly enhanced gelling capacity: up to a 4.4-fold increase in gel firmness and a 3.5-fold increase in gel elasticity over untreated protein. Microstructural examination showed small oil droplets dispersed in a densely packed gel matrix when MP was oxidatively modified, and the TGase treatment further contributed to such packing. Comparison of the modification of MP via GluOx oxidation/TGase cross-linking pathway under different salt concentrations (0.3 and 0.6 M NaCl) showed different patterns of MP cross-linking, resulting in different extents of aggregation. Under low-salt condition (0.3 M NaCl), the GluOx/TGase treatment increased the gel strength to the same level as those treated with TGase in 0.6 M NaCl, suggesting a potential application of GluOx/TGase for improving gel strength in low ionic strength conditions. Finally, the application of GluOx oxidation in the development of emulsion-type sausages was studied. The GluOx oxidation/TGase cross-linking improved the textural properties (firmness, chewiness, and rupture force) of emulsion-type sausages under both salt levels (P < 0.05). Under low-salt condition (1.5% NaCl), GluOx/TGase treatment can increase the sausage binding strength to the same level as the control sample under high-salt condition (3% NaCl). The GluOx oxidation/TGase treatment shows promise to improve the textural properties of emulsified meat products. However, the significant decrease of a* value and increase of b* value indicated GluOx-induced color deterioration.

<p>Aerobic exercise can stimulate mixed muscle protein synthesis (MPS) acutely post-exercise; however, the types of proteins synthesized as a result of aerobic exercise are not known by studying changes in mixed MPS. We aimed to study the effect of aerobic exercise intensity on the 4 and 24 h post-exercise fractional synthesis rate (FSR) of myofibrillar proteins. Using a within-subject design, eight males (21 ± 1 years, VO_{2 peak}: 46.7 ± 2.0 mL kg^-1 min^-1) underwent 2 trials with a primed constant infusion of L-[<em>ring</em>-¹³C₆]phenylalanine in the fasted state for each work-matched exercise intensity (LOW: cycling for 60 min at 30% W_max and HIGH: 30 min at 60% W_max). Muscle biopsies were obtained to determine resting, 4 and 24 h post-exercise myofibrillar FSR. We also studied the phosphorylation of signaling proteins involved in protein synthesis at each time point using immunoblotting methods. Phospho-p38^{Thr180/Tyr182} was greater at 4.5 h after exercise compared to 0.5, 24 and 28 h post-exercise (<em>p</em> < 0.05). Additionally, a strong trend was present for phospho-mTOR^Ser2448 (<em>p</em> = 0.056) with 0.5 h post-exercise phosphorylation significantly higher after HIGH than after LOW exercise (<em>p </em>< 0.05). Myofibrillar protein synthesis was stimulated 1.5–fold 0.5 – 4 h post-exercise (<em>p</em> < 0.05), returning to rest in the LOW condition 24 h post-exercise, while 6 out of 8 subjects maintained increased myofibrillar FSR 24 h post HIGH exercise (<em>p</em> < 0.05). The increase in myofibrillar FSR 0.5 – 4 h post-exercise was correlated with phospho-mTOR^Ser2448 0.5 h post-exercise (r = 0.698, <em>p</em> < 0.01), indicating the role of this signaling pathway in myofibrillar protein synthesis. It is concluded that aerobic exercise has an effect on myofibrillar protein synthesis and intensity may play a role in the duration of this response.</p> / Master of Science in Kinesiology